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Biochip substrate and biochip

a technology of biochips and substrates, applied in the field of biochips, can solve the problems of high cost, difficult stabilization and synthesis of long oligonucleotides, and variability in the amount of immobilized molecules

Active Publication Date: 2017-10-03
HIPEP LAB +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The substrate design prevents spot diffusion and cross-contamination, enabling efficient and uniform immobilization of molecules, increasing the detection sensitivity and capacity, and allowing for the use of smaller sample volumes while maintaining detectable binding reactions.

Problems solved by technology

Among the above-mentioned two methods, the Affymetrix method has a drawback in that stable immobilization and synthesis of a long oligonucleotide are difficult because the oligonucleotide is synthesized on the surface of the substrate, and that the cost is also high.
However, it is known that since these functional groups or the polylysine are attached to the entire surface of the substrate, spots may be diffused, cross-contamination may occur due to contacts between adjacent spots, and the amounts of the immobilized molecules may differ when some spotting methods are used.
However, the amount of the molecules which can be bound by the modification of the silanol groups on the surface of the glass is small, and when a slide glass, a generally used substrate, is used, the amount is several nanomoles, so that the capacity of the substrate to immobilize the molecules is low.
In case of immobilizing the molecules by adsorption, there is also a drawback in that non-specific adsorption strongly occurs, so that the fluorescent substances in unreacted areas, which remain even after washing after the biological reactions, decrease the S / N ratio of the detection.

Method used

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  • Biochip substrate and biochip
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  • Biochip substrate and biochip

Examples

Experimental program
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Effect test

example 1

1. Production of Substrate for Biochips (Part 1)

[0025]A high purity Al—Mg alloy plate (Mg content: 4% by weight) with a thickness of 1.2 mm was sized to 26 mm×76 mm by punching with a press. A plurality of the plates were stacked and annealed under pressure under an atmosphere at 340° C., thereby removing strain and attaining a flatness of not more than 5 μm. Thereafter, working of the end faces and chamfer (specifically, angle 45°, a length: 0.2 mm) was performed to prepare plates with a size of 25 mm×75 mm. Then each plate was ground with a double side grinding machine 16B produced by SpeedFam, in which a sponge grindstone was mounted, to attain a thickness of 0.98 mm and a degree of parallelization of not more than 1 μm. Then micro recesses with a size shown in FIG. 1 were formed in the substrate by mechanical processing with a microdrill using a marking press CAMM-3 produced by Roland. The resulting plate was then subjected to, in the order mentioned, degreasing, etching, acid a...

example 2

2. Production of Substrate for Biochips (Part 2)

[0027]After placing a thermosetting phenol resin in a mold, a two-step heat treatment at 90° C. and 120° C. was performed to prepare a Bakelite block. A plate having a thickness of 2 mm and a size of 31 mm×95 mm was cut out from the block, and was ground with a double side polishing machine 16B produced by SpeedFam, in which an iron surface plate was mounted, to attain a thickness of 1.30 mm and a degree of parallelization of not more than 1 μm. After chamfering, the resulting plate was slowly heated to 1200° C. thereby carbonizing the substrate to amorphous carbon. Thereafter, using a LD-excited YVO4 laser produced by Fuji Electric, the small recesses having the size shown in FIG. 1 were formed in the air, and then functional groups were attached by the following method: First, the substrate was set in a stainless steel vessel having a window made of a synthetic quartz, and irradiated with an ultraviolet lamp (lamp output power: 110 W...

example 3

[0028]Calmodulin was measured using a peptide chip having recesses in which a peptide having α-helix structure was immobilized, which peptide was labeled with different fluorescence labels at its both ends. More specifically, this was carried out as follows:

[0029]The sequence of the core region of the peptide having α-helix structure was designed by molecular modeling using a computer (molecular modeling using Insight II / Discover of Molecular Simulation, U.S.) based on the amino acid sequence of the peptide described in a reference (K. T. O'Neil and W. F. DeGrado, Trend Biochem Sci, 15, 59-64 (1990)). As a result, the designed amino acid sequence of the core region was Leu-Lys-Lys-Leu-Leu-Lys-Leu-Leu-Lys-Lys-Leu-Leu-Lys-Leu (SEQ ID NO:1). This sequence is known to specifically bind to calmodulin. To this sequence, a Cys residue as an anchor for immobilization, and as fluorescently labeled residues, Lys(TAMRA) and Lys(FAM) were added. Thus, an amino acid sequence Cys-Lys(TAMRA)-Leu-L...

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Abstract

A biochip substrate which is free from cross-contamination due to spot spreading or contact with spots adjacent to each other, and a biochip using the same. A biochip substrate on which multiple valleys for immobilizing biological substances are formed so as to prevent cross-contamination due to spot spreading or contact with spots adjacent to each other, and a biochip using the same are provided. Moreover, it is found out that a desired binding in a target molecule contained in a test sample occurs at a detectable level in a solution system even in the case where a valley have such a small capacity as 1 nL to 10 nL.

Description

TECHNICAL FIELD[0001]The present invention relates to a biochip on which a biologically relevant substance(s) such as nucleic acids, peptides, sugars and the like is(are) immobilized, as well as to a substrate therefor.BACKGROUND ART[0002]It is well known that biochips having a flat substrate surface on which DNAs or proteins are immobilized include those prepared by Affymetrix method in which oligonucleotides are synthesized on the surface of the substrate using photolithography, and those prepared by Stanford method in which preliminarily provided probe DNAs or probe proteins are spotted so as to immobilize them on the surface of the substrate. Either type of the biochips is used such that fluorescence is detected after biological reactions with a target, and identification of the molecule or diagnosis is performed from the resulted pattern.[0003]Among the above-mentioned two methods, the Affymetrix method has a drawback in that stable immobilization and synthesis of a long oligon...

Claims

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Application Information

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Patent Type & Authority Patents(United States)
IPC IPC(8): C40B60/14G01N33/543B82Y30/00B01L3/00
CPCG01N33/54393B01L3/5085B82Y30/00B01L2200/12B01L2300/0636B01L2300/0829B01L2300/0893B01L2300/0896B01L2300/12C40B60/14
Inventor NOKIHARA, KIYOSHIOKA, YASUO
Owner HIPEP LAB